EFFECTS OF CHANGES IN SOME SOLVENT PARAMETERS ON CARBOXYPEPTIDASE A IN SOLUTION AND IN CROSS-LINKED CRYSTALS

Abstract

Quiocho and Richards1 have shown that oii treatmeilt with the bifunctional reagent glutaraldehyde, single crystals of the enzyme carboxypeptidase A become completely insoluble. The effect appears to be due to intermolecular cross-linking which provides ani effectively infinite three-dimensional4network. The material gives single crystal X-ray diffraction patterns very similar to those obtained with the untreated protein. Both the native and cross-linked crystals have demonstrable catalytic activity for hydrolysis of the protected dipepticle substrate carbobenzoxyglycyl-L-phenylalanine (CGP). More recent studies have begun to define the enzymic properties of both the crystalline and amorphous enzyme.2 Access of substrate (CG1P) to the interior of the crystals is not limited by diffusion if the minimum crystal dimension is 5-10 t or less. The substrate concentration required to reach half-maximal activity is roughly the same for all forms of the enzyme. The shapes of the velocity versus substrate concentration curves for the solid samples of the enzyme differ from those in solution, but they are still indicative of multiple substrate-binding sites. In passing from the soluble enzyme to very small native crystals at low ionic strength the specific activity measured with CG1' decreases by a factor of about 300. The cross-linking reaction per se results in a further decrease inactivity of onily a factor of 2.